The Mixed Lineage Leukemia (MLL) gene codes for a histone methyltransferase that is frequently mutated by chromosomal translocations in human leukemias associated with a poor clinical outcome. The studies proposed in this competitive renewal application address the hypothesis that the leukemogenic actions of MLL oncoproteins are critically dependent on proteins recently discovered to associate with MLL or its fusion partners, and that the activities or interactions of these associated factors constitute potential targets for molecular therapies. This hypothesis is based on major discoveries during the current award period that have substantially advanced our understanding of the molecular mechanisms of MLL leukemias. We have shown that menin, a tumor suppressor protein and MLL- associated factor, is paradoxically required for MLL-mediated leukemogenesis. Menin functions as a transcriptional co-factor for MLL oncoproteins, and in this capacity we demonstrated that its only role is to promote interactions with a newly discovered MLL- associated protein known as LEDGF/p75. The latter is a chromatin-associated protein with co-activator activity but unknown molecular function, previously implicated in leukemia and HIV pathogenesis. Studies in the first specific aim will characterize the molecular functions of LEDGF/p75 required for MLL-mediated leukemogenesis using biochemical and genetic approaches in pre-clinical and cell line transformation model systems. These studies will identify a potential ligand for the putative methyl-lysine recognition motif of LEDGF/p75 and also establish the feasibility of antagonizing their interactions as a molecular therapeutic strategy. Studies in the second aim will employ genetic and biochemical techniques to determine the molecular mechanisms that underlie oncogenic activation of LEDGF/p75 itself by chromosomal translocations in leukemias, and establish its specific roles in leukemogenesis mediated through the MLL and/or Myc transcriptional pathways. Studies in the third specific aim are based on our discovery of a multi-protein complex (AEP) that contains several MLL fusion partners and the P-TEFb transcription elongation factor. This novel complex is tethered by a subset of MLL oncoproteins to critical target genes in MLL leukemia cells, however its molecular contributions required for leukemogenesis have not been fully defined. Our studies will further characterize the AEP complex, its critical enzymatic activities, its implications for MLL transcriptional regulation in general, and its role in MLL-mediated leukemogenesis. The therapeutic value of targeting the activities or interactions of AEP components with pathogenic roles in MLL leukemia will be interrogated using preclinical transformation models.